A Breeder's guide to Techniques and Services

What is embryo transfer?

Strictly speaking the term ‘embryo transfer’ refers solely to the collection of an embryo from a donor animal and its subsequent transfer into the uterus of a recipient. However, by common usage, it has become accepted to cover a whole range of allied techniques, including superovulation, freezing, splitting, sexing etc.

Day –12 insert PRID/CIDR

Day –4 inject prostaglandin

Day –2 remove PRID/CIDR

Day 0 reference heat

Day 10 FSH treatment starts

Day 13 inject prostaglandin

Day 15 heat & insemination

Day 22 uterine flush

+ insert PRID/CIDR

+ inject prostaglandin

Day 32 remove PRID/CIDR

Day 34/35 oestrus

Superovulation

Under normal circumstances a cow that is healthy, open and cycling regularly will ovulate and release one egg from her ovaries when she comes into heat. If she is inseminated the single embryo can be collected (flushed) and transferred, but in most situations a more cost effective approach is to treat the cow with a course of hormone (FSH) injections which stimulates the ovaries and induces multiple ovulations. This process is called ‘superovulation’, and an example programme is shown in Fig 1. Our routine treatment schedule consists of twice daily FSH injections for 4 days although other regimes are applied from time to time, depending on circumstances.

The degree of response is related to the number of small follicles which are naturally present in the cow’s ovaries. This number can vary dramatically between individual animals which accounts for the variability in response that we experience (see ‘results’ section later). The ovarian follicle population can be influenced significantly by management (see our management advice leaflet). The dose, treatment regime and type of FSH can also affect the size of response. In fact some regimes appear to suit particular cows, and this can only be found out by trial and error!

Occasionally cows ‘over-respond’ to treatment and too many follicles are stimulated. Over-response is undesirable as it can often result in poor ovulation, poor fertilisation or poor recovery of eggs. Some cows and some breeds are more sensitive than average to the FSH, and dose adjustments are necessary to achieve satisfactory results.

Fig 2. Bull Sperm

Insemination

A standard dose of young bull semen contains about 20 million sperm. After insemination most of the sperm are lost in the uterus and only about 100 complete the journey to the part of the reproductive tract where fertilisation takes place.

In fact there is strong evidence that in superovulated cows the raised circulating hormone levels interfere with sperm transport and even fewer sperm are present. When a bull becomes popular the situation can be made worse because the number of sperm per straw is often progressively reduced below the standard 20 million. This is not necessarily a problem for routine AI because careful monitoring of non-return rates mean herd fertility should not suffer.

However, the combination of less sperm and more eggs to fertilise in an ET programme puts the whole process on a knife edge and makes attention to detail at insemination more important.

Fertilisation failure is a real problem in ET programmes But a number of things can be done to tip the balance in our favour:

Wherever possible use semen from bulls of known high fertility. Discuss your choice with us—we usually know which bulls are performing well.

Timing of insemination is important and the time of onset of heat can vary from our scheduled programme. We usually recommend 2 inseminations with a total of 3 straws of semen. The first insemination (2 straws) should ideally be about 12 hours after the onset of standing heat; the second (1 straw) about 12-18 hours later. Discuss timing with us if in doubt.

Good, clean AI technique is important. Rough handling or incorrect semen deposition e.g. part way through the cervix, are not conducive to good results.

In some cases where there is doubt over semen fertility it can be beneficial to use 2 bulls to inseminate the donor. Fertilisation rate can be better than when either bull is used alone and the calves can easily be differentiated by DNA typing.

Natural service can be used but, surprisingly, is not always as effective as AI. If frozen semen is available from the bull in question, the best results are achieved by combining natural service and AI.

Of course, semen quality and AI technique & timing are not the only factors influencing fertilisation:

Age of donor—old cows (over 12 years) are generally less productive and poor fertilisation is often the reason.

Disease—e.g. mastitis or lameness can cause significant stress, resulting in poor fertilisation. Conditions such as these should be treated before an ET programme starts.

Embryo Collection

Is normally carried out 7 days after heat and takes about 30 minutes per cow. An epidural (local anaesthetic into the tail head) is administered to reduce straining and discomfort during the flush. A long PVC catheter is then manipulated into the uterine horn and held in position by inflating a small balloon cuff, which also makes a fluid tight seal for the part of the uterus to be flushed (Fig 3).

The catheter has 3 passages—one is used to inflate the cuff, a second to inject flushing fluid into the uterus and a third is connected to a filter which collects the flushing fluid after it has washed the uterus, trapping the embryos and debris. Manipulation of the horn during the flush ensures a thorough washing of the uterus and an efficient embryo collection. The process is repeated for both uterine horns and the contents of the filter are tipped into a special dish for searching under a microscope. As the embryos are located in among the debris they are picked out and placed in a special holding medium. A farm office or kitchen can function as a temporary ET laboratory for microscope work (Fig 5).

The recovered embryos are examined under high power and classified viable, degenerate or unfertilised. The viable embryos are then graded for quality (1,2 or 3—good, moderate or poor) and loaded into straws for transfer or freezing.

Embryos are held on the bench in a nutrient fluid which maintains their full viability for at least 8 hours, allowing time consuming techniques such as sexing to be carried out with confidence.

Typically normal fertile donors will yield an average of around 5 embryos per flush, with a range of 0-30 or more, but the same factors that affect response will also affect recovery. Young cows (less than 10 years old), with no history of ill health or sub-fertility are the best and most consistent donors. Maiden heifers, older cows (over 10 years old) or those with a poor breeding history, perform more inconsistently, averaging fewer viable embryos.

Fig 3. Diagram showing catheter in place for flushing

Fig 4. A flush of 7 day old embryos

Fig 5. Our laboratory at Newbiggin

Freezing Embryos

Embryos can either be transferred into heat synchronised recipients on the same day as collection, or frozen and stored for transfer at a later date. Freezing is now routine technology and involves washing the embryo thoroughly, to remove any viral or bacterial contamination, prior to equilibrating with a biological antifreeze solution and loading into labelled straws. Slow and precise cooling to sub-zero temperatures in a programmable freezing machine then allows the straws to be stored in liquid nitrogen indefinitely.

In some cases the use of a microscope is necessary during thawing (Fig 5), but a relatively new development in freezing technology allows thawing to take place beside the recipient and direct transfer without use of a microscope. The majority of embryos are now frozen for direct transfer.

Over large numbers of transfers pregnancy rate with frozen/thawed embryos averages about 56% (about 10% lower than after fresh transfer). Although embryos from the majority of cows will freeze successfully, there can be variation between donors with a small minority producing poor results.

Splitting Embryos

Good quality embryos can be divided or ‘split’, with each half embryo having the potential to develop into a calf. The procedure is carried out using a special inverted microscope and a micromanipulator with micro scalpel blade attached (Figs 6 & 7), but can be easily carried out ‘on-farm’.

Half embryos are usually transferred into separate recipients, but both halves can be transferred into one recipient, giving the possibility of twins. Twins will, of course, be genetically identical and same sex.

Typically, half embryos transferred singly will yield a pregnancy rate of 50-55%, (i.e. 100%+ pregnancy rate per embryo), providing a significant increase in number of calves produced per embryo.

Fig 6. A split embryo - the dark shape is the scalpel

Fig 7. Embryo sexing and splitting can be done on-farm

Sexing Embryos

Sex can be determined by using the splitting equipment to slice a small piece (biopsy) off the embryo which is then run through DNA fingerprinting process to detect the presence of a male chromosome. Briefly, the biopsy is added to a small tube containing the sexing reagents and placed into a thermocycler for about 1½ hours. During this period the minute amount of target DNA is multiplied sufficiently to test and binds with a fluorescent dye in the tube to give a pink colour when viewed under ultra-violet light (Fig 8). The male embryos can then be identified and picked out. The test is virtually 100% accurate for males and over 95% accurate for females, i.e. the occasional bull calf will be born when transferring ‘female’ embryos.

The sexing procedure does not adversely affect pregnancy rates when embryos are transferred fresh. A double abuse however, such as sexing and freezing, does reduce pregnancy rates and we normally recommend synchronising some recipients for fresh transfer.

Use of Sexed Semen

The ideal way to produce embryos of the desired sex would be to use sexed semen to inseminate the donor. Unfortunately sexed semen is not generally of good enough quality to use in ET programmes and the fertilisation rate is usually poor. Consequently we are not yet recommending its use.

Fig 8. The pink tubes contain biopsies from male embryos

Day 0 Insert CIDR

Day 7 Inject prostaglandin

Day 8 Remove CIDR

Day 9-11 Detect and record heat

Day 17 Embryo transfer

Fig 10. Crossbred recipients with calves at foot

Recipient synchronisation and transfer of embryos

For advice on selection and management of recipients see our ET Management advice booklet.

Fresh or frozen-thawed embryos can be transferred into suitable recipients that are at the same stage of the heat cycle as the donor at embryo collection. Using the onset of standing heat as a reference point, animals in heat within 24 hours before or after the donor are suitably synchronised. Heat synchronisation can be achieved using prostaglandin injections alone (single or double 11-14 days apart), or preferably by combining prostaglandin and insertion of a progesterone releasing intravaginal device such as a PRID or CIDR (Fig 9).

Accurate heat detection is an important part of an ET programme (see our ET management notes).

All recipients are examined prior to transfer and any that are unsuitable for transfer are rejected. Common reasons for rejection include ovarian cysts, inadequate corpus luteum and ovulation failure. Rejection rates are usually higher at times of weather or nutritional stress but most of the problems are temporary and should not affect future fertility of the recipient.

The transfer itself is more technical than, but similar to AI. In brief, the embryo is loaded into a plastic straw which, in turn is loaded into a transfer gun. The recipient is prepared with an epidural anaesthetic and the technician manipulates the gun per rectum through the cervix and as far as possible up the uterine horn on the same side as the corpus luteum before depositing the embryo.

Results can vary according to circumstances but, on average, embryos transferred fresh yield a pregnancy rate of 65%, falling to 56% after freezing. Many factors can affect pregnancy rate after transfer, including:

Nutritional status—It is very important to have recipients on a rising plane of nutrition and well supplemented with trace elements (see recipient management notes).

Body condition—Recipients should be maintaining or increasing body weight, but not be over fat. Animals losing condition for whatever reason, are likely to give poor results.

Embryo quality—Embryos are classified into 3 quality grades, good moderate and poor and these grades correlate well with pregnancy rate. Poor embryos have a low chance of creating a pregnancy but if they survive, they produce perfectly normal calves.

Age of recipient—Maiden heifers will generally give 5-10% better pregnancy rate compared to cows, however, in some situations this can be countered by the improved calving ease of cows.

Disease status—Infectious diseases such as BVD, IBR and Leptospirosis can have a disastrous effect on an ET programme and control measures should be discussed with your Vet.

It is inevitable that some losses will also occur after positive pregnancy diagnosis. A resorption/abortion rate of 5% from 6 weeks is considered normal. Most losses occur early in pregnancy and are spontaneous, however, it is wise to investigate known abortions in case the cause is infectious.

Running an ET Programme

Planning: Programmes take some time to organise and you should allow 6-8 weeks before the flush, or longer if recipients are to be purchased. Several items require attending to:

Nutritional management of donor cows and recipients, including trace element bolusing, should be planned and initiated.

All routine vaccinations and treatments should be completed by 6 weeks pre-flush/transfer.

Organise semen.

Organise and inform your AI technician service of your intentions.

Check with the breed society regarding ET regulations.

If necessary, organise handling facilities for flush and transfer. For instance, it should be possible to handle animals under cover if the weather is bad.

Liaise with your veterinary surgeon over programme details.

A donor reference heat programme will be forwarded to you 4-5 weeks before the projected day of ET, and provided the donor behaves normally and shows a good heat this will be followed by a combined donor superovulation and recipient synchronisation programme (see fig 1 and fig 9). The programmes detail the action dates and times necessary to achieve a successful result and will be copied to your veterinary surgeon as his/her input will be required.

Facilities required: Donors can be flushed in a simple crush or AI stall, as long as there is room behind the donor for 2 people and a small table. Some protection from the weather is desirable. Ideal handling facilities for recipients are a race and crush, although locking yokes work well if the cattle are used to them.

The microscope for embryo searching etc can be set up on a table or desk in a farm kitchen or office (see fig 6 and fig 7).

After the flush: To bring the donor back into a normal cycle we treat her with a CIDR and prostaglandin (see fig 1). She should be monitored carefully for heat signs after CIDR removal. If a good heat is not seen within 5 days she may need additional treatment and should be examined by your veterinary surgeon. If a good heat is seen, however, she can either be served to go in calf or the heat can be used as a reference point for a repeat flush.

DNA typing: All breed societies require donor cows to have a DNA type on record. We normally take a hair sample at the time of flush to fulfil this requirement.

Registering embryos and ET calves: After the flush we will register your embryos with the relevant breed society and forward a copy of the paper work to you, together with a summary report of the work done. The embryo registration form should be retained as the unique form number must be quoted when registering the resulting calves with the breed society.